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Issued  April  22, 1913. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— Bulletin  166. 


A.  D.  MELVIN,  Chief  of  Bureau. 


^.lEMICAL  CHANGES  PRODUCED  IN  COWS' 

MILK  BY  PASTEURIZAip^j^^  j^Y 


of  California 
1  Regional 
^  Facility 


AUG  7    1913 


BY 


PHILIP  RUPP.  M.  D.,  Ph.  D., 

Chemist,  Dairy  Division. 


\\\ 


WASHINGTON: 
GOVERNMENT  PRINTING  OFFICE. 

1"13. 


Digitized  by  the  Internet  Archive 

in  2007  with  funding  from 

IVIicrosoft  Corporation 


http://www.archive.org/details/chemicalchangesOOruppiala 


Issued  April  22, 1913. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  166. 

A.  D.  MELVIN,  Chief  op  Bureau. 


CHEMICAL  CHANGES  PRODUCED  IN  COWS' 
MILK  BY  PASTEURIZATION. 


BY 
PHILIP  RUPP,  M.  D.,  Ph.  D. 

Chemist,  Dairy  Division. 


WASHINGTON: 
GOVERNMENr  PRINTING  OFFICE. 

1913. 


BUREAU  OF  ANIMAL  INDUSTRY. 


Chief:  A.  D.  MELvm. 

Assistant  Chief:  A.  M.  Farrington. 

Chief  Cleric:  Charles  C.  Carroll. 

Animal  Hiisbandry  Division:  George  M.  Rommel,  chief. 

Biochemic  Division:  M.  Dorset,  chief. 

Dairy  Division:  B.  H.  Rawl,  chief. 

Field  Inspection  Division:  R.  A.  Ramsay,  chief. 

Meat  Inspection  Division:  R.  P.  Steddom,  chief. 

Pathological  Division:  John  R.  Mohi.er,  chief. 

Quarantine  Division:  Richard  W.  Hickman,  chief. 

Zoological  Division:  B.  H.  Ransom,  chief. 

Experiment  Station:  E.  C.  Schroeder,  superintendent. 

Editor:  James  M.  Pickens. 

DAIRY  DIVISION. 
B.  H.  Rawl,  Chief. 

Helmer  Rabild,  in  charge  of  Dairy  Farming  Investigations. 
S.  C.  Thompson,  in  charge  of  Dairy  Manufacturing  Investigations. 
L.  A.  Rogers,  in  charge  of  Research  Laboratories. 
Ernest  Kelly,  in  charge  of  Market  Milk  Investigations. 
Robert  McAdam,  in  charge  of  Renovated  Butter  Inspection. 
2 


LETTER  OF   TRANSMITTAL. 


U.  S.  Department  of  Agricultitre, 

Bureau  of  Animal  Industry, 

Washington,  D.  C,  January  30,  1913. 
Sir:  I  have  the  honor  to  transmit  for  piihlication  as  a  bulletin 
of  this  bureau  the  accompanying  manuscript,  entitled  "Chemical 
Changes  Produced  in  Cows'  Milk  by  Pasteurization,"  by  Dr.  Philip 
Rupp,  chemist  in  the  Dairy  Di\'ision  of  tliis  bureau.  The  paper  de- 
scribes a  series  of  experiments  made  with  the  object  of  determining 
the  effect  of  pasteurization  at  various  temperatures  on  the  chemical 
composition  of  cows'  milk.  The  work  was  undertaken  because  of 
certain  ol)jections  which  are  made  against  pasteurization,  it  having 
been  asserted  that  the  process  injures  the  milk  from  a  digestive  and 
nutritive  stand})oint,  particular^  as  a  food  for  infants.  It  is  satis- 
factor}'"  to  note  from  the  results  of  the  work  that,  so  far  as  the  ordi- 
nary temperatures  used  in  commercial  pasteurization  are  concerned, 
these  objections  are  not  well  founded. 

The  method  of  filtering  the  milk  necessary  for  the  carr^^Tiig  on  of 
the  tests  consists  of  a  new  ap])lication  by  Dr.  Hupp  of  the  clay -cell 
fdtration  used  by  other  scientists.  This  method  is  fully  described 
and  illustrated  by  a  diagram. 

Respectfully,  A.  D.  Melvin, 

(Idef  of  Ihireau. 
Hon.  James  Wilson, 

Secretary  of  Agriculture . 

80002°— Bull.  lf.<>— 13  3 


CONTENTS. 


Page. 

Introduction ." 5 

Method  of  pasteurization  employed 5 

Chemical  changes  in  the  phosphates 6 

Method  of  filtration 6 

Methods  of  analysis 7 

Changes  in  the  composition  of  the  serum  during  filtration. 8 

Chemical  changes  in  the  proteins 11 

Lactalbumin 11 

Methods  of  analysis 11 

Casein 13 

Method  of  coagulation 14 

Changes  in  the  acidity 15 

Summary  and  conclxisions 15 


ILLUSTRATION. 


Pase. 
Fig.  1.  Apparatus  for  filtering  milk 7 

4 


CHEMICAL  CHANGES  PRODUCED  IN  COWS'  MILK  BY  PASTEURIZATION. 


INTRODUCTION. 

One  of  the  objections  frequently  raised  against  pasteurized  milk 
is  that  the  heating  produces  changes  in  the  chemical  composition 
which  make  it  unsuitable  for  infant  nourishment.  It  is  claimed  that 
a  portion  of  the  soluble  phosphate  of  lime  becomes  insoluble  and  that 
this  change  produces  defective  nutrition,  while  the  changes  in  the 
lactalbumin  and  in  the  casein  render  the  pasteurized  milk  more 
indigestible  than  raw  milk,  and  therefore  inferior  as  a  food.  These 
objections  were  brought  forward  at  a  time  when  high  temperatures 
were  employed  in  the  sterihzation  of  infants'  milk,  and  they  are  made 
use  of  at  the  present  day  by  the  opponents  of  pasteurization,  even 
though  the  temperatures  used  in  pasteurizing  are  considerably  lower. 

The  objects  of  the  work  herein  described  were  to  study  the  chemical 
changes  produced  by  the  heating  of  milk  at  the  different  temperatures 
used  in  commercial  pasteurization  and  to  determine  whether  a  tem- 
perature that  destroys  all  pathogenic  germs  can  be  used  in  pasteuri- 
zation without  having  any  appreciable  effect  on  the  chemical  compo- 
sition of  the  milk.  Tlu*ee  points  are  considered:  First,  the  changes 
in  the  phosphates  are  discussed  in  order  to  show  to  what  extent  the 
soluble  phosphate  of  lime  becomes  insoluble.  Second,  the  proteins 
are  examined  to  determine  to  what  extent  the  albumin  is  precipi- 
tated and  the  casein  changed  in  its  characteristic  property  of  coagu- 
lating with  rennin.     Finally,  the  acidity  of  the  milk  is  considered. 

METHOD  OF  PASTEURIZATION  EMPLOYED. 

Tlie  milk  was  pasteurized  in  a  constant-temperature  bath.  The 
water  having  reached  the  required  temperature,  the  cool  milk,  in  an 
Erlenmeyer  flask  closed  with  a  valve  to  prevent  evaporation,  was 
placed  in  the  water  and  occasionally  moved  about  until  it  had  ac- 
quired the  temperature  of  the  water.  This  usuaUy  required  from 
15  to  20  minutes,  according  to  the  height  of  the  temperature.  It 
was  then  allowed  to  remain  for  30  minutes,  after  which  it  was  rapidly 
cooled  to  about  12°  C.  (53°  F.).  The  milk  used  was  of  very  good 
quality  and  had  a  very  low  bacterial  count.  The  variation  in  tem- 
perature during  pasteurization  was  less  than  0.5°  C.  (0.9°  F.). 

5 


6  CHEMICAL  CHANGES  PRODUCED  BY  PASTEURIZATION. 

CHEMICAL  CHANGES  IN  THE  PHOSPHATES. 

The  vast  majority  of  the  experiments  which  show  a  diminution  in 
the  quantity  of  soluble  calcium  salts  in  heated  milk  as  compared  with 
raw  milk  refer  to  milk  which  has  been  heated  to  the  boiling  point. 
But  few  definite  results  can  be  found  showing  a  diminution  of  the 
soluble  calcium  phosphate  in  pasteuriized  milk. 

DifBoth  ^  found  a  decrease  in  the  soluble  phosphates,  amounting  to 
a  loss  of  25.9  per  cent,  on  heatmg  milk  to  60°  C.  (140°  F.)  for  30 
minutes,  while  Babcock  ^  believes  that  a  change  in  the  solubility  of  the 
lime  salts  is  brought  about  when  the  milk  is  pasteurized,  and  bases 
his  conclusion  on  the  less  ready  coagulabUity  of  the  milk  by  rennet. 

In  order  to  determine  the  effect  of  pasteurization  on  the  solubihty 
of  the  phosphates  in  the  mUk,  the  serum  of  both  raw  and  pasteurized 
milk  was  examined. 

METHOD   OF   FILTRATION. 

A  new  application  of  the  clay-cell  filtration  as  used  by  Soxhlet ' 
was  employed.  The  clay  cell  of  a  galvanic  element  is  thoroughly 
cleansed  by  boiling  in  dilute  caustic  soda  and  then  washed  by  filter- 
ing hot  water  through  the  cell  by  suction.  The  cell  is  next  boiled  in 
dilute  hydrochloric  acid  and  again  washed  by  suction  with  hot  dis- 
tilled water  until  the  filtrate  no  longer  gives  a  reaction  for  chlorin. 

The  method  of  filtration  is  easily  understood  by  a  description  of 
the  accompanying  diagram  (figure  1). 

An  inverted  bell  jar  (a)  (9  inches  high  and  4  inches  in  diameter)  is 
covered  with  a  perforated  glass  plate  (6)  holding  a  tube  containing 
a  plug  of  cotton  to  admit  air.  Inside  the  jar  is  a  porous  clay  cell  (c) 
of  a  galvanic  element  (5^  inches  high,  2J  inches  in  diameter,  and  the 
cell  wall  one-eighth  of  an  inch  thick)  the  opening  of  which  is  covered 
with  a  piece  of  rubber  dam  (d)  of  medium  thickness  and  supported  on 
a  heavy  glass  plate  (e).  Both  the  rubber  dam  and  the  glass  plate  are 
perforated  for  the  passage  of  a  rubber  stopper  containing  a  glass  tube 
with  a  Geissler  stopcock  (/).  The  upper  end  of  the  tube  is  flush  with 
the  glass  plate  e,  while  the  lower  end  passes  into  a  filtering  flask  (g) 
of  750  c.  c.  capacity.  A  glass  tube  with  a  two-way  stopcock  (h)  is 
attached  to  the  outlet  of  the  flask,  and  this  in  turn  is  connected  with 
the  suction  apparatus  by  means  of  a  heavy  rubber  tube.  In  order  to 
obtain  airtight  joints  a  little  stopcock  grease  is  placed  on  the  glass 
plate,  and  the  edge  of  the  clay  cell  is  also  coated  with  a  thin  layer. 
The  cover  (6)  of  the  bell  jar  is  held  down  in  the  same  manner. 

•  Diffloth,  Paul.  Du  r61e  de  quelqucs  agents  physiques  et  chimiques  dans  I'insolubilisat  ion  des  phosphates 
du  lait.  Bulletin  des  Sciences  Pharmacologiques,  vol.  10,  p.  273-279.  Paris,  1904.  Abstract:  Zeitschrift 
fCir  Untersuchung  dcr  Nahrungs-  und  Genussmittel,  vol.  11,  no.  7,  p.  455-456.    Berlin,  Apr.  15,  1906. 

»  Babcock,  S.  M.  The  centrifugal  septaration  of  casein  and  insoluble  phosphates  from  milk.  Wisconsin 
Agricultural  Exi)eriment  Station,  Twelfth  Annual  Report  (1895),  p.  93-99.    Madison,  1896. 

*  Soxhlet,  Franz.  Beitriige  zur  physiologischen  Chemie  der  Milch.  Journal  fur  Praktische  Chemie, 
new  series,  vol.  6,  no.  1/2,  p.  1-52.    Leipsic,  Aug.  24, 1873. 


CHEMICAL   CHANGES  IN    THE   PHOSPHATES. 


To  begin  filtration  remove  the  cover,  evacuate  the  apparatus,  and 
while  the  suction  is  still  on  fill  the  bell-jar  with  milk.  Then  cut  off 
the  suction  at  h,  replace  the  cover,  and  remove  the  suction  tube.  The 
apparatus  is  now  ready  to  be  placed  in  the  refrigerator. 

The  advantage  of  this  method  over  those  previously  employed  is 
that  the  filtration  can  be  continued  indefinitely  and  the  serum 
removed  at  any  time  without  interfering  with  the  filtration.  Further- 
more, no  concentration  of  the  serum  can  take  place,  as  evaporation  is 
excluded.  The  serum  can  be  removed  at  any  time  by  closing  the 
stopcock  /,  admitting  air  by  opening  Ji,  j, 

and  removing  the  filtering  flask.     Then    '  ,         tji?^ 
replace  the  flask,  evacuate,  close  the  stop-  ^^ 

cock  Ti,  and  open/,  and  the  apparatus  is 
ready  for  further  filtration. 

METHODS    OF    ANALYSIS. 

Weigh  about  40  grams  of  milk  (equiva- 
lent to  about  0.1  gram  of  phosphoric 
acid),  evaporate  to  dryness,  and  ignite 
at  a  low  red  heat  until  the  ash  is  free  from 
carbon.  Dissolve  the  ash  in  hot  water 
containing  a  little  hyilrochloric  acid  and 
dilute  to  about  100  c.  c. 

Basic  acetate  precipitation.  —Almost 
neutralize  the  solution  of  the  asli  with 
ammonia,  add  2  c.  c.  of  dilute  acetic  acid 
and  a  slight  excess  of  ferric  <'hlorid  solu- 
tion of  known  strength  (7.5  c.  c.  of  a  10 
per  cent  solution).  Now  add  13  to  14 
c.  c.  of  a  10  per  cent  ammonium  acetate 
solution,  dilute  to  850  to  400  <•.  c.  and 
boil  for  about  one-half  to  one  minute, 
stirring  occasionally  to  j)revoiit  bumping. 
If  the  quantity  of  ferric  chlorid  nddtxl  was 
sullicient  the  ])rc<-ipitate  will  be  brownish- 
red,  and  the  j)recipitatioii  will  bo  complete  as  the  boiling  point  is 
reached  if  the  acetate  was  adih^l  in  suflicient  <iunntity.  Filter  boil- 
ing hot  through  two  12.5  cm.  filters  and  wash  immediatidy  with  a  hot 
dilute  ammonium  acetate  solution  (one-half  to  one  \)ov  cent),  usiiig  a 
line  jet  whi(di  is  played  around  tlie  edge  of  the  precijutate,  thus  cut- 
ting it  free  from  the  pa|)er  in  order  to  produce  rapid  filtration.  Do 
not  allow  to  drain  dry.  Wash  the  precipitate  in  tliis  maiiner  three 
to  four  times  until  free  from  <'hlorids,  return  to  the  original  beaker 
with  hot  water,  and  dissolve  1)V  the  addition  of  nitric  acid.     When 


FiCi.  1      Ajiiiaratus  for  fi lining  milk. 


8  CHEMICAL  CHANGES  PBODUCED  BY  PASTEURIZATION. 

the  basic  acetate  precipitation  is  carried  out  as  described  above  the 
filtration  is  always  rapid  and  the  precipitate  never  runs  through  the 
filter.  In  washing  the  precipitate  with  hot  water,  some  flakes  gen- 
erally appear  on  concentrating  the  filtrate  and  washings.  This, 
however,  is  not  the  case  if  a  hot  dilute  solution  of  ammonium  acetate 
is  used. 

The  phosphoric  acid  in  the  basic  acetate  precipitate  was  determined 
by  the  molybdate  method  and  weighed  as  magnesium  pyrophosphate. 
The  filtrate  and  washings  from  the  basic  acetate  precipitation  were 
concentrated  and  the  calcium  precipitated  with  ammonium  oxalate. 
The  oxalate  was  redissolved,  reprecipitated,  and  weighed  as  calcium 
oxid.  The  filtrate  and  washings  from  calcium  precipitation  were 
evaporated,  ammonium  salts  removed,  and  the  magnesium  deter- 
mined as  pyrophosphate. 

For  serum  analysis  evaporate  about  70  grams  and  ignite  at  a  dull 
red  heat  untU  the  ash  is  free  from  carbon.  In  the  basic  acetate  pre- 
cipitation use  6  c.  c.  of  ferric  chlorid  solution  and  9  to  10  c.  c.  of 
ammonium  acetate  solution. 

The  casein  phosphorus  was  determined  by  Neumann's  method,* 
using,  however,  only  10  c.  c.  of  concentrated  sulphuric  acid.  The 
total  phosphorus  in  the  milk  was  also  determined  by  Neumann's 
method. 

The  specific  gravity  was  taken  by  means  of  a  picnometer. 

CHANGES    IN    THE    COMPOSITION    OF   THE    SERUM   DURING   FILTRATION. 

De  Vries  and  Boekhout,^  using  a  Chamberland  filter  and  pressure, 
foimd  that  the  quantity  of  calcium  in  the  serum  did  not  vary  in  differ- 
ent fractions  during  the  filtration,  the  first  20  c.  c.  of  serum  containing 
the  same  quantity  of  calcium  as  the  second  and  third.  In  order  to 
determine  whether  the  serum  remains  of  constant  composition  during 
filtration  and  for  what  length  of  time  milk  can  be  filtered  at  a  tem- 
perature of  5°  to  8°  C.  (41°  to  46.  4°  F.)  without  changing  its  composi- 
tion, skim  milk  was  filtered  and  the  serum  analyzed  during  a  period 
of  two  weeks.  Skim  milk  was  used  for  this  experiment  because  it 
filters  more  rapidly  than  whole  milk.  The  results  are  shown  in 
Table  1. 

1  Neumann,  Albert.  Einfache  Veraschungsmethode  (Sauregenusch-Veraschung).  Zeitschrlft  fttr 
Physlologische  Chemie,  vol.  37,  no.  2,  p.  115-142.    Strasburg,  Dec.  20, 1902. 

*  Ott  de  Vries,  J.  J.,  and  Boekhout,  F.  W.  J.  Beltrag  zur  Kenntnis  der  Labgerinnung.  Die  Land- 
wlrtachaftlichen  Versuchs-Stationen,  vol.  55,  no.  3,  p.  221-239.    Berlin,  Feb.  12, 1901. 


CHEMICAL  CHANGES  IN   THE  PHOSPHATES. 


9 


Table  1. — Quantity  of  soluble  phosphoruLs  in  the  serum  on  different  days  during  the 
filtration  of  raw  skim  milk,  separated  at  30°  C.  {86^  F.). 


Quantity. 


Sp)eciflc 
gravity. 


Acidity.' 


PjO,. 


CaO. 


MgO. 


Casein 

Raw  skim  milk 

Serum: 

First  day — 3  hours 

First  day— next  18  hours 

Second  day 

Do 

Third  day 

Fourth  day 

Fifth  day 

Sixth  and  seventh  days 

Do 

Tenth  and  eleventh  days 

Thirteenth  and  fourteenth  days 

Fifteenth  and  part  of  sixteenth  day 


Orams. 


34.3 

81.5 
109.8 


1.034 

1.023 
1.025 
1.025 


9.0 


109.3 
112.0 
105.7 
205.0 


1.025 
1.025 
1.025 
1.025 


9.0 
9.0 
9.0 
9.0 


155.0 
1.5S.5 
105. 0 


1.025 
1.025 


10.0 
12.0 
14.6 


Per  f^nt. 

0.0638 

.2352 

.0753 
.1029 
.1054 
.1055 
.1055 
.1048 
.1054 
.1057 
.  105.3 
.1074 
.1082 
.1110 


Per  cent. 


Per  cent. 


0. 1671 

.0450 
.0554 
.0581 
.0588 
.0578 
.0585 
.0589 
.0579 
.0582 
.0609 
.0633 
.0683 


0.0185 

.0133 
.0133 
.0134 
.0133 
.0127 
.0127 
.0130 
.0129 
.0131 
.0131 
.0139 
.0145 


'  Number  of  cubic  centimeters  of       alkali  required  to  neutralize  100  c.  c.  of  serum. 

As  will  be  seen  from  the  above  table  there  is  an  absorption  of  both 
phosphoric  acid  and  calcium  at  the  beginning  of  filtration,  while  the 
magnesium  passes  completely  through  the  cell  wall.  For  this  reason 
the  first  portion  of  the  serum  must  be  rejected  as  worthless  for  making 
comparisons.  The  quantity  to  be  rejected  varies  with  the  size  of  the 
cell  used  and  may  amount  to  from  60  to  90  c.  c. 

The  specific  gravity  of  the  serum  collected  during  the  first  3  hours 
was  1.023,  and  it  increased  to  1.025  during  the  next  18  hours,  after 
which  time  it  remained  constant  during  the  remainder  of  the  experi- 
ment. 

The  acidity  was  8.8  on  tlie  fii"st  tlay  and  9  on  the  second  day.  After 
the  ninth  day  the  acidity  began  to  increase  gradually,  being  10  on  the 
tenth  and  eleventh  days,  while  on  the  fifteenth  day  it  was  14.6,  showing 
that  the  lactic  acid  had  considerably  increased. 

The  phosphoric  acid  during  the  firet  3  houra  amounted  to  only 
0.0753  per  cent,  and  it  increased  to  0.1029  per  cent  during  the  next 
18  hours.  On  the  second  day  it  was  0.1054  per  cent,  and  then  re- 
mained constant  until  the  tenth  and  eleventh  days,  when  it  began 
gradually  to  increase,  amounting  to  0.1 1 10  per  cent  on  the  fifteenth  day. 

The  calcium  content  was  also  low  during  the  first  3  hours  of  filtra- 
tion, amounting  to  0.045  per  cent,  and  it  increased  to  0.0554  per  cent 
during  the  ne.xt  18  hours.  On  the  second  day  it  was  0.0581  per  cent, 
and  began  to  increase  gradually  as  the  acidity  increased,  amounting 
to  0.0683  per  cent  on  the  fift(^enth  day. 

The  magnesium  remained  c»)nstant  during  the  first  11  days  and 
then  increased  very  slowly  u])  to  the  fifteenth  day. 

The  experiment  shows,  furthermore,  that  the  filtration  of  a  clean 
milk  with  low  bacterial  count  can  be  continued  for  about  one  week 
without  any  appreciable  chemical  changes  taking  j)lace  in  the  serum, 
provided  the  temperature  be  kej)!  at  6°  to  8^  C.  (42.8°  to  46.4°  F.). 


10 


CHEMICAL  CHANGES   PRODUCED  BY   PASTEURIZATION. 


Before  proceeding  to  the  filtration  of  raw  milk  in  the  refrigerator, 
several  experiments  were  made  in  which  formaldehyde  had  been 
added  to  the  milk  as  a  preservative.  This  was  done  in  order  to 
carry  on  the  filtration  at  room  temperature.  The  results  obtained 
showed  that  tVe  serum,  after  the  first  day's  filtration,  remained  of 
constant  composition,  and  led  up  to  carrying  out  the  filtration  of  a 
pure  milk  hi  the  refrigerator. 

Having  thus  shown  that  the  serum  remains  of  constant  composi- 
tion after  the  first  day  of  filtration,  the  next  step  was  to  determine 
whether  there  were  any  demonstrable  changes  produced  in  the 
soluble  phosphates  by  pasteurization.  In  order  to  produce  a  maxi- 
mum precipitation  the  highest  temperature,  68.3°  C.  (155°  F.), 
employed  in  the  holder  process  was  chosen.  The  results  obtained 
are  shown  in  Table  2. 

Table  2. — Quantity  of  soluble  phosphates  irt  the  serum  of  raw  whole  milk  and  in  whole 
milk  pasteurized  at  68.3°  C.  (155°  F.)for  SO  minutes. 


Specific 
gravity. 

Acidity. 

PsOo. 

CaO. 

MgO. 

Casein 

Per  cent. 

0.0595 

.2219 

.     .  1074 
.1078 
.1082 

.1078 
.1063 
.1089 

Per  cent. 

Percent. 

Raw  whole  milk 

1.029 
1.026 

18.0 
9.8 

0. 1749 

.0625 
.0624 
.0631 

.0611 
.0613 
.0627 

0  0203 

Raw  serum: 

Second  and  third  days 

0142 

Do 

0142 

Fifth  and  sixth  days 

1.026 
1.026 

9.6 
9.6 

0143 

Pasteurized  senmi: 

Second  and  third  davs 

0140 

Do ". 

.0140 

Fifth  and  sixth  days 

1.026 

9.4 

0141 

It  will  be  seen  from  the  table  that  the  changes  produced  by 
pasteurization  at  68.3°  C.  (155°  F.)  for  30  minutes  are  trifling.  The 
phosphoric  acid  content  remains  the  same  in  both  the  raw  and  the 
pasteurized  serum.  The  quantity  of  lime  salts,  while  a  trifle  lower 
in  the  pasteurized  than  in  the  raw  serum,  is  still  within  the  limit  of 
error,  and  the  magnesia  remains  the  same  in  both  serums. 

Other  experiments  made  with  skim  milk  showed  results  agreeing 
in  every  particular  with  this  one  made  with  whole  milk.  All  prelim- 
inary experiments  were  made  with  skim  milk  because,  as  previously 
stated,  it  filters  more  readily  and  thus  gives  a  larger  quantity  of 
serum  for  analysis. 

Comparing  Table  1  with  Table  2  we  find  that  the  variation  in  the 
quantity  of  soluble  lime  salts  between  the  two  samples  of  milk  is 
far  greater  than  that  found  between  the  raw  and  pasteurized  seiTims. 
For  this  reason  the  objection  frequently  raised  against  pasteurized 
milk  as  an  infant  food,  on  the  ground  that  the  soluble  lime  salts  are 
diminished  by  the  process  of  pasteurization,  is  without  foundation. 


CHEMICAL  CHANGES  IN   THE   PROTEINS.  11 

CHEMICAL    CHANGES   IN   THE    PROTEINS. 
LACTALBUMIN. 

Different  investigators  do  not  agree  as  to  the  exact  temperature 
at  which  the  coagulation  of  the  albumin  begins.  Steiner  '  found  no 
change  on  heating  milk  for  25  minutes  at  60°  C.  (140°  F.),  while 
Solomin  ^  says  that  the  change  seems  to  begin  at  this  temperature, 
and  Jensen  and  Plattner  ^  also  found  some  coagulation  in  milk 
heated  for  five  houi-s  at  60°  C.  (140°  F.).  Babcock  *  could  find  no 
change  on  heating  milk  for  20  minutes  at  65°  G.  (149°  F.),  while 
Woll  ^  states  that  coagulation  begins  at  this  temperature.  According 
to  Willoughby  « the  change  begins  at  70°  C.  (158°  F.),  while  de  Freud- 
enreich  '  found  15  to  20  per  cent  coagulated  on  heating  milk  for 
30  mmutes  at  68°  to  69.5°  C.  (154.4°  to  157°  F.).  A  series  of  experi- 
ments were  therefore  undertaken  to  determine  at  what  temperature 
the  change  in  the  albumin  begins,  and  to  what  extent  it  is  coagulated 
at  the.  different  temperatures  commonly  employed  in  the  pasteuriza- 
tion of  milk. 

METHODS   OF   ANALYSIS. 

Casein. — To  10  grams  of  milk  were  added  50  c.  c.  of  distDled  water 
at  40°  C.  (104°  F.),  then  1.5  to  2  c.  c.  of  a  saturated  alum  solution 
were  added  gradually.  The  j)rccipitate  was  allowed  to  settle,  was 
then  filtered  and  washed.  The  precipitate  and  filter  ])aper  wore 
treated  by  the  Gunning  method,  adding  about  0.2  gram  of  copper 
sulphate. 

Albumin  and  globulin. — The  albumin  and  globulin  in  the  filtrate 
and  washings  were  precipitated  with  10  c.  c.  of  Alm6n's  tannin  solu- 
tion; this  was  allowed  to  settle,  was  then  filtered  and  washed.  The 
precipitate  was  treated  the  same  as  under  casein. 

Tables  3  to  6  show  the  results  of  the  experiments  to  determine  the 
lactalbumin  precipitated  in  whole  milk  pasteurized  for  30  minutes  at 
temperatures  ranging  irom  02.8°  C.  (145°  F.)  to  71.1°  C.  (100  F.). 

'  Steiner,  U.  licit riige zur  Kcnntnia  dcs  Kinflus-sos  der  I'asteiirisicrung  auf  die  Ucsohadcnheit  dcr  Milch 
und  auf  den  Buttcnings  I'roze^sz.  Milch-Zeituii},',  vol.  30,  no.  20,  p.  401-4a'{,  June  29;  no.  2S,  p.  435,  July  13. 
Leipsic,  1901. 

'Solomin,  1'.  Uebcr  die  beiin  Krliilzen  der  MiK'h  ausfallendon  EiweLssniongcn.  .Vrchiv  fiir  Hygiene, 
vol.  2S,  no.  1,  p.  4:MS.     Municli  and  Leipsic,  IS'Hi. 

»  Jensen,  Orla,  and  Plattner,  Krnesl.  De  ractioii  du  chauffago  .sur  le  lait  de  vaelie.  Upvhc  (loncrale  dii 
Lait,  vol.  4,  no.  ir.,  p.  3iil-3(W,  .May  :«);  no.  IT,  ]>.  3^^J97,  Juno  15;  no.  IS,  p.  419-424.     I.ierre,  UK15. 

*  Babcock,  S.  M.  The  centrifugal  separation  of  c;\sein  and  insoluble  ph(xsphate,s  from  milk.  \VL>^-onsin 
Agricultural  Experiment  Station,  Twelfth  .Vtunial  Keport  (IS95),  p.  9:5-99.    Madi.son,  ls9t'i. 

5  Woll,  F.  V>.  The  efTei^t  of  pa.stourization  and  sterilization  on  the  vLsccxsity  and  fat  globulus  of  milk 
and  cream.  Wiscon.sin  .\gricultur;d  Experiment  Station,  Twelfth  .Vnnual  Uc|K)rt  (l>S95t,  p.  104-173. 
Madi-son,  189«\ 

«  Willoughby,  Edward  F.    Milk,  it.s  production  and  u.se,s.     lA)ndon,  19();<.    .Sec  p.  12.")-120. 

'  He  Freudenreich,  Ed.  Stir  l;i  piv^teurisation  du  lait  dans  ralimentatiou  de  I'eufance.  Hevue  (icnirale 
du  Lait,  vol.  4,  no.  19,  p.  433-J37.    Lierro,  July  15,  1905. 


12 


CHEMICAL  CHANGES  PRODUCED  BY  PASTEURIZATION, 


Table  3. — Quantity  of  albumin  precipitated  in  raw  whole  milk  and  in  milk  pasteurized 
at  62.8°  C.  {145°  F.)for  SO  minutes. 


Raw  milk. 

Pasteurized  millc. 

I. 

n. 

Average. 

I. 

II. 

Average. 

Casein  nitrogen     .  . 

Per  cerU. 

0.4003 

.0996 

Per  cerU. 

0.4027 

.0998 

Per  cent. 

0.4015 

.0997 

Per  cent. 

0.4015 

.0996 

Percent. 

0.4010 

.0997 

Per  cent. 
0  4013 

0997 

PROTEIN- NITR0GENX6. 38. 


Casein 

AJbumin+globulin 


2.554 
.635 


2.569 
.637 


2.562 
.636 


2.561 
.635 


2.558 
.636 


2.560 
.636 


No  albumin  precipitated. 

Table  4. — Quaniiiy  of  albumin  precipitated  in  raw  whole  milk  and  in  milk  pasteurized 
at  65.6°  C.  (160°  F.)for  SO  minutes. 


RawmOk. 

Pasteurized  milk. 

I. 

n. 

Average. 

I. 

II. 

Average. 

Per  cent. 

0.4260 

.0983 

Per  cent. 

0.4218 

.0978 

Per  cent. 

0.4239 

.0981 

Per  cent. 

0.4320 

.0920 

Per  cent. 

0.4328 

.0929 

Per  cent. 
0  4324 

.0925 

PROTEIN=NITROGENX6. 38. 

Casein                      

2.717 
.627 

2.691 
.624 

2.704 
.626 

2.756 
.587 

2  762            2  759 

AJbumin+globulin 

.593               .'>on 

Albumin  precipitated— 5. 75  per  cent. 

Table  5. — Quantity  of  albumin  precipitated  in  raw  whole  milk  and  in  milk  pasteurized 
at  6S.S°  C.  {155°  F.)for  SO  minutes. 


Raw  milk. 

Pasteurized  milk. 

I. 

II. 

Average. 

I. 

II. 

Average. 

Per  cent. 

0.3739 

.0963 

Per  cent. 

0.3812 

.0981 

Per  cent. 

0.3776 

.0972 

Per  cent. 

0.3910 

.0863 

Percent. 

0.3878 

.0833 

PercetU. 
0.3894 

Albumin+ globulin  nitrogen 

.0848 

PROTEIN- NITROGENX6.38. 


Casein 

Albumin + globulin  , 


2.385 
.614 


2.432 
.626 


2.409 
.620 


2.495 
.551 


2.474 
.531 


2.486 
.541 


Albumin  precipitated— 12.75  per  cent. 


CHEMICAL  CHANGES  IN   THE   PROTEINS. 


13 


Table  6. — Qtuintity  of  albumin  precipitated  in  raw  whole  milk  and  in  milk  pasteurized 
at  71.1°  C.  (160°  F.)for  SO  minutes. 


Raw  milk. 

Pasteurized  milk. 

I. 

II. 

Average. 

I. 

11. 

Average. 

Per  cent. 

0.38(J0 

.0961 

Per  cent. 

0.3851 

.0989 

Per  cent. 

0.3856 

.0975 

Per  cent. 

0.4200 

.0681 

Per  cent. 

0.4169 

.0667 

Per  cent. 
0.4185 

.0674 

PROTEIN=  NITROGENX6.38. 

Casein                             

2.462 
.613 

2.457 
.631 

2.460 

.622 

2.679 
.434 

2.660 
.426 

2.670 

.430 

Albumin  precipitated—  30.87  per  cent. 

From  an  analysis  of  the  above  tables  it  is  apparent  that  no  albumin 
is  coagulated  on  heating  milk  for  30  minutes  at  62.8°  C.  (145°  F.), 
the  temperature  most  commonly  used  in  the  holder  process  in  com- 
mercial pasteurization.  At  65.6°  C.  (150°  F.)  the  separation  of  the 
albumin  has  begun,  5.71  per  cent  havmg  become  insoluble.  At  68.3° 
C.  (155°  F.)  the  quantity  has  increased  to  12.76  per  cent,  wnile  at 
71.1°  C.  (160°  F.)  30.87  per  cent  of  the  albumin  has  been  coagulated. 

CASEIN. 

The  condition  in  which  casein  exists  in  milk  has  not  as  yet  been 
fully  explained.  The  effect  of  heat  shows  itself  in  two  ways.  In  the 
first  place,  the  rennin  coagulation  may  be  accelerated,  retarded,  or 
even  inhibited;  and  in  the  second  place,  the  coagulum  may  assume 
a  different  character  from  that  obtained  from  raw  milk. 

The  great  majority  of  authors  agree  that  no  change  in  rennin 
coagulation  takes  place  below  65°  C.  (149°  F.).  Steiner  *  found  a 
retardation  in  milk  heated  to  60°  C.  (140°  F.),  while  Fleischmann  and 
Morgen  ^  found  the  milk  less  sensitive  to  rennin  when  heated  for  two 
hours  from  60°  to  70°  C.  (140°  to  158°  F.).  According  to  Van  Slyke 
and  Publow  ''  milk  heated  above  65.6°  C.  (150°  F.)  for  a  considerable 
length  of  time  coagulates  less  rapidly  and  the  coagulum  is  highly 
flocculent.  De  Freudenreich  *  finds  the  coagulum  unchanged  at  68° 
to  60.5°  C.  (154.4°  to  157.1°  F.).  Jensen  and  Plattner  '  state  that 
retardation  begins  on  heating  milk  for  five  hours  at  70°  C.  (158°  F.), 
while  Mayer  *  i)laces  the  beginning  at  75°  C.  (167°  F.).     Stassano  and 

'  Loo.  cit. 

«  Fleischmann,  W.,  and  MorRcn,  A.  Einipes  iilxT  die  nach  SchorfT's  Vorfahrrn  conscrvirto  Flaschcn 
milch.     Die  Landwirths<'hafllichon  Vcrsuch  Stationoii,  vol.  28.  p.  321  332.    Berlin,  1883. 

•  Van  Slyke,  Lucius  L.,  and  Publow,  Charles  .\.  The  science  and  practice  of  cheese-making,  p.  310. 
New  York,  1909. 

♦  Mayer,  Adolf.  Bestimmungen  der  Wirksamkeit  des  Labfermentos  unter  verschiedenen  auszeren  Um- 
Sttoden.  Milch-Zeitung,  vol.  10, no.  2,  p.  17-19,  Jan.  12;  no.  3,  p.  33-38,  Jan.  19;  no.  4,  p.  49-52,  Jan.  26;  no. 
6,  p.  81-84,  Feb.  9.    Bremen,  1881. 


14 


CHEMICAL  CHANGES  PRODUCED  BY  PASTEURIZATION. 


Talarico  *  differ  from  all  the  preceding  in  that  thej  observed  an  accel- 
eration of  rennin  coagulation  in  milk  heated  from  55°  to  65°  C.  (131° 
to  149°  F.),  while  at  70°  C.  (158°  F.)  the  heated  milk  behaved  the 
same  as  raw  milk,  and  above  70°  C.  (158°  F.)  the  retardation  began. 
The  following  experiments  were  made  with  raw  milk  and  milk 
pasteurized  at  various  temperatures: 


METHOD   OF   COAGULATION. 


The  apparatus  consisted  of  a  large  galvanized-iron  box  having 
four  cylindrical  compartments  surrounded  by  water.  The  upper 
openings  were  of  sufficient  size  to  admit  a  cylindrical  percolater 
(1  foot  high  and  2^  inches  in  diameter),  the  lower  openings  being 
about  one-fourth  of  an  inch  in  diameter.  A  small  glass  tube  drawn 
out  into  a  capillary  was  inserted  into  the  neck  of  the  percolator  by 
means  of  a  perforated  rubber  stopper. 

In  making  the  test,  the  water  in  the  box  was  heated  to  the  required 
temperature,  the  percolators  were  then  placed  in  the  compartments 
and  allowed  to  remain  untU  they  had  acquired  the  temperature  of 
the  bath,  the  upper  openings  being  covered.  Next  200  c.  c.  of  milk 
were  heated,  the  rennin  solution  added,  and,  after  mixing,  poured 
into  the  percolator.  The  upper  opening  was  then  closed  with  a  cover 
and  the  time  required  for  the  milk  to  coagulate  in  the  tube  noted. 
The  flow  through  the  tube  was  regulated  by  breaking  off  the  capillary 
to  such  an  extent  that  aU  tubes  dropped  at  the  same  rate. 

For  each  experiment  200  c.  c.  of  milk  were  heated  in  an  Erlenmeyer 
flask  to  35°  C.  (95°  F.)  and  5  c.  c.  of  rennin  solution  (0.15:  100  c.  c. 
of  water)  added.  This  was  taken  as  the  beginning  of  the  coagula- 
tion time;  the  end  point  was  when  the  milk  stopped  dropping  from 
the  capillary  tube.  The  rennin  solution  was  made  of  sucli  strength 
that  the  raw  milk  coagulated  in  about  20  minutes.  The  results  are 
shown  in  the  following  table: 

Table  7. —  Time  required  for  rennin  coagulation  of  raw  whole  milk  and  of  whole  milk 
pasteurized  at  different  temperatures.  Milk  200  c.  c,  rennin  solution  {0.15  gram: 
100  c.  c.  of  water)  5  c.  c. 


Experiment. 


Raw 
milk. 


Milk  pasteurized  at- 


55"  C. 
(131°  F.). 


60°  C.    65°  C. 
(140°  F.;.  a49°  F.). 


70°  C. 
(158°  F.). 


75"  C. 
(167°  F.). 


Af.  g. 

18  30 

18  08 

19  34 
19  23 


Af.   8. 

17  28 
16  56 


M.  t. 

17  10 
16  53 


M.  s. 

17  12 
17  12 


M.   3. 


M.     3. 


20  38 
20  25 


36  30 

37  30 


'  Stassano,  H.,  and  Talarico,  J.  De  I'inflflence dc  lacuisson  sur  la  casf^ificationdu  lait  par  le  lab-ferment. 
Comptes  Rendus  Hebdomadaires  des  Stances  et  M^moires  de  la  Soci^t^  de  Biologie,  vol.  69  (annte  62, 
tome  2),  no.  28,  p.  254-255.    Pan ;,  Aug.  5, 1910. 


SUMMARY  AND  CONCLUSIONS.  15 

From  the  above  table  it  will  be  seen  that  milk  pasteurized  at  55°  C. 
(131°  F.),  60°  C.  (140°  F.),  and  65°  C.  (149°  F.)  all  coagulate 
more  rapidly  with  rennin  than  does  the  raw  milk.  This  result  was 
obtained  in  all  cases  in  a  large  number  of  experiments.  At  70°  C. 
(158°  F.)  the  retardation  of  rennin  coagulation  has  begun,  while  at 
75-°  C.  (167°  F.)  the  time  has  about  doubled  and  tlie  coagulum  is 
higlily  flocculent. 

CHANGES  IN  THE  ACIDITY. 

Woll '  and  de  Freudenreich  ^  find  no  appreciable  change  in  the 
acidity  of  milk  pasteurized  at  67°  to  68°  C.  (153°  to  154.4°  F.)  for 
20  minutes.  Hoft^  and  Jensen  and  Plattncr  '  on  the  other  hand 
find  a  diminution  in  tlie  acidity  on  heating  milk. 

From  a  large  number  of  experiments  made  during  the  coagulation 
of  milk  by  remiin,  it  was  found  that  the  acidity  of  the  milk  alwa3's 
diminished  during  pasteurization.  While  tlie  diminution  was  slight 
it  amounted  to  several  tenths  of  a  cubic  centimeter,  and  was  always 
sufficient  to  be  determined  by  titration  with  tenth-normal  caustic- 
soda  solution.  Tlic  cUminution,  liowever,  is  insignificant  and  is 
probably  due  to  a  loss  of  carbonic  acid  during  the  heating.  Further- 
more, the  diminution  varies  in  different  samples  of  milk. 

SUMMARY  AND  CONCLUSIONS. 

1.  Milk  pasteurized  by  the  hohler  process  at  62.8°  C.  (145°  F.)  for 
30  minutes  does  not  undergo  any  appreciable  chemical  cliange. 

2.  The  soluble  phospliates  of  lime  and  magnesia  do  not  become 
insoluble.  At  68.3°  C.  (155°  F.)  the  quantity  of  phosphoric  acid, 
Ume,  and  magnesia  in  the  serum  of  botli  raw  and  pasteurized  milk 
are  practically  the  same. 

3.  Tlic  albumin  does  not  coagulate  at  62.8°  C.  (145°  F.),  but  at 
65.6°  C.  (150°  F.)  5.75  per  cent  of  the  albumin  is  rendered  insoluble. 
As  the  tem})erature  increases  the  amount  of  coagulated  albumin 
increases.  At  68.3°  C.  (155°  F.)  the  quantity  increases  to  12.75  per 
cent,  and  at  71.1°  C.  (160°  F.)  it  amounts  to  30.78  })er  cent. 

4.  The  time  required  for  coagulating  the  casein  by  rennin  is  slightly 
less  in  milk  pasteurized  at  tem])eratures  up  to  (>5°(\  (14!)°  F.)  than  it 
is  in  raw  milk.  At  70°  C.  (15S°  F.)  there  is  a  slight  retardation, 
while  at  75°  C.  (167°  F.)  the  time  lias  almost  doubled. 

5.  The  acidity  as  determined  by  titration  is  slightly  diminished 
in  pasteurized  milk. 

I  I.oc.  c'it. 

»  Moft,  11.     rel)or  dio  \criinilcriiiif;  dor  .\ciditiit  dor  Milch  IxMiii  Krhiizcn.     Mikh-Zoiiung,  vol.  :50.  no. 
7,  p.  10:f.     I.oipzig,  Kob.  Ki,  l<Jf)l. 

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